Department of Chemistry and Optical Science and Technology Center, University of Iowa, Iowa City, IA 52242, USA.
Proc Natl Acad Sci U S A. 2010 Oct 19;107(42):17974-9. doi: 10.1073/pnas.0912190107. Epub 2010 Sep 27.
The potential for femtosecond to picosecond time-scale motions to influence the rate of the intrinsic chemical step in enzyme-catalyzed reactions is a source of significant controversy. Among the central challenges in resolving this controversy is the difficulty of experimentally characterizing thermally activated motions at this time scale in functionally relevant enzyme complexes. We report a series of measurements to address this problem using two-dimensional infrared spectroscopy to characterize the time scales of active-site motions in complexes of formate dehydrogenase with the transition-state-analog inhibitor azide (N(3)(-)). We observe that the frequency-frequency time correlation functions (FFCF) for the ternary complexes with NAD(+) and NADH decay completely with slow time constants of 3.2 ps and 4.6 ps, respectively. This result suggests that in the vicinity of the transition state, the active-site enzyme structure samples a narrow and relatively rigid conformational distribution indicating that the transition-state structure is well organized for the reaction. In contrast, for the binary complex, we observe a significant static contribution to the FFCF similar to what is seen in other enzymes, indicating the presence of the slow motions that occur on time scales longer than our measurement window.
飞秒到皮秒时间尺度的运动影响酶催化反应固有化学步骤速率的潜力是一个存在重大争议的来源。在解决这一争议的核心挑战中，难以在功能相关的酶复合物中实验表征此时间尺度的热激活运动。我们报告了一系列使用二维红外光谱进行的测量，以解决该问题，用于在甲酸脱氢酶与过渡态类似物抑制剂叠氮化物（N(3)(-))的复合物中表征活性部位运动的时间尺度。我们观察到具有 NAD(+)和 NADH 的三元复合物的频-频时间相关函数（FFCF）以缓慢的时间常数 3.2 ps 和 4.6 ps 完全衰减。这一结果表明，在过渡态附近，活性部位酶结构以窄且相对刚性的构象分布进行采样，表明过渡态结构为反应组织良好。相比之下，对于二元复合物，我们观察到与在其他酶中看到的类似的 FFCF 中的显著静态贡献，表明存在比我们的测量窗口更长时间尺度上的缓慢运动。
